The invention relates to a temperature-regulated cell perifusion chamber for use in microscopy, cell imaging, cell culture and cell biology.
The development of new techniques in cell culture has successfully led to the growing of pure lines of many types of mammalian cells. However, exploitation of the availability of new pure cell cultures is impeded by the lack of appropriate instrumentation. Specifically, it is currently difficult or impossible by commercial instruments to continuously observe mammalian cells under an optical microscope at high power, while at the same, time, controlling temperature and altering it as appropriate, and maintaining the composition and sterility of a defined growth medium or altering it as appropriate.
One method of studying the intracellular composition of cells is by means of fluorescence imaging. In this system, a narrow laser beam is directed through a microscope onto cells attached to a glass surface. Fluorescence within the cells induced by the laser is digitally recorded, amplified by a photomultiplier fixed to the microscope, and analyzed by computer. By loading cells with specialized commercially available fluorescence dyes one can study changes in concentrations of intracellular sodium, potassium, calcium, and hydrogen ions, as well as membrane voltage and membrane fluidity, and almost any other material to which a specific antibody can be produced and labeled with a fluorescence dye. For this method to be successful, the cells must be firmly attached to a transparent surface and remain immobile during the entire experimental procedure. Furthermore, the temperature of the medium bathing the cells must be absolutely controlled.
An important type of experiment is to determine the effects of various pharmacological agents, electrolytes, nutrients and environment on the composition, interactions and structure of cells and subcellular compartments. In order to carry out such studies, it is necessary to fix the cell to a transparent glass or Pyrex surface, take fluorescent measurements and change the composition of the medium bathing the cells with a new solution containing the compound under study while constantly or periodically recording the fluorescence. While a few cell types adhere tightly to a glass surface and are conveniently studied, many cell types, among the most interesting and important, do not adhere strongly to glass. In a conventional cell chamber used in such studies, the physical process of removing the old solution (aspiration) and exchanging it with a new one (pipetting) detaches the cells, causing them to move and often to float away so that the fluorescence measurements are useless. Furthermore, it is difficult to maintain rigid temperature control in such chambers.
Conventional cell perifusion chambers have one or more of the following disadvantages: are manufactured of metals which may partially deteriorate under acidic or alkaline conditions leading to toxic activity toward cells, may alter optical path by assembly by the uneven tightening of screws, may be difficult to clean, have slow rates of temperature change and are of heavy mass, composed of many parts which must be cleaned and assembled each time, may not have the capacity for thermoregulation, or the cell chamber is unenclosed and open to the atmosphere and not providing protection against pathogens.
Thus, there is a need for a Cell Perifusion Chamber:
1. that is of durable construction so that it may be used with little regard to mechanical failure under harsh conditions,
2. That is constructed of biocompatible material to permit normal cell growth,
3. that is of low mass to prevent distortions in the optical path on a microscope and can be used in space science in a low cost in mass,
4. that can withstand heat, acid and alkali so that it can be sterilized by several methods,
5. that can be temperature-controlled within ranges appropriate for living cells,
6. in which the temperature can be changed rapidly,
7. that can permit exchange of solutions (perifusion) which is so gentle that the cell is not disturbed and remains unmoved on its glass surface,
8. that can be used with a standard 35mm microscope stage insert,
9. that can be completely sealed to safely observe pathogens and other hazardous materials for prolonged periods of time,
10. that is of simple design permitting easy and rapid assembly and disassembly for cleaning and sterilization,
11. in which the optical path through the cell is uniform and not distorted significantly by variations in sealing pressure during assembly.
An object of the present invention is to fulfill the need referred to above. In accordance with the principles of the preset invention, this objective is obtained by providing a temperature-regulated cell perifusion chamber that permits the observation of cells under a microscope at high power (e.g., 1000 xc3x97), over a prolonged period of time (hours to weeks), is suitable for fluorescence imaging experiments, while permitting the simultaneous changing of the medium bathing the cells, maintaining the sterility of the medium and absolutely controlling temperature.
The cell perifusion chamber structure includes a cell chamber body having a support surface with an aperture defined through the support surface, and wall structure extending upwardly from the support surface to define an interior. The wall structure includes passages therein. A gasket is disposed on the support surface so as not to cover the aperture. A first transparent cover is disposed on the gasket so as to cover the aperture. A water bath body is provided and has a first portion and a second portion extending from the first portion. The first portion defines a second support surface. The second portion is received in the interior of the cell chamber body and is in interference fit arrangement with the wall structure. The water bath body has an interior support surface with an aperture therethrough. The aperture extends through the first and second portions. The first portion has first ports therein which communicate with the aperture of the water bath body. The second portion includes second ports therein which communicate with the aperture of the water bath body and with associated passages in the cell chamber body. A second transparent cover is disposed as on the interior support surface of the water bath body so as to divide the aperture of the water bath body into first and second portions. The second transparent cover covers the second portion of the aperture to define a sealed cell chamber enclosed by the second transparent cover, the first transparent cover and surfaces of the cell chamber body. A transparent window is disposed on the second support surface to cover the first portion of the aperture to define a water bath chamber enclosed by the transparent window, the second cover and surfaces of the water bath body. Fluid may enter and exit the water bath chamber via first ports to regulate temperature of the cell chamber via heat exchange therewith, and perifusion fluid may enter and exit the cell chamber via the second ports.
In accordance with another aspect of the invention, a method of regulating temperature of a cell perifusion chamber includes:
providing a sealed cell chamber and a transparent cover in the cell chamber, the transparent cover supporting cells to be studied,
providing a water bath chamber defined by surfaces which are in heat exchange relation with surfaces defining the cell chamber, the water, bath chamber being fluidly isolated from the cell chamber,
supplying temperature regulating fluid flow through the cell chamber so as to regulate a temperature of the cell chamber, and
supplying perifusion fluid flow to through the cell chamber.